Effect of Kisspeptin-Type Neuropeptide on Locomotor Behavior and Muscle Physiology in the Sea Cucumber Apostichopus japonicus

Simple Summary The sea cucumber Apostichopus japonicus is an important aquatic invertebrate, which has high nutritional and medicinal value. Kisspeptins are neuropeptides encoded by the kiss1 gene, and little is known about them outside of the vertebrate lineage. In this study, we investigated the effect of KPs on locomotor behavior in one control group and two treatment groups (AjK1 and AjK2). We found that AjK1 had a significant dose effect, mainly by reducing the stride length and duration of movement to decrease the movement distance of sea cucumbers, whereas AjK2 had little inhibitory effect at the same dose. The levels of phosphatidylethanolamine (PE), phosphatidylcholine (PC),uridine, glycine, and L-serine in the longitudinal muscle of A. japonicus treated with AjK1 differed significantly from those of the control, which may explain the observed changes in locomotor behavior. Abstract Kisspeptins are neuropeptides encoded by the kiss1 gene, and little is known about them outside the vertebrate lineage. Two kisspeptin-type neuropeptides (KPs) have been discovered in Apostichopus japonicus (AjK1 and AjK2), an edible sea cucumber, and have been linked to reproductive and metabolic regulation. In this study, we evaluated how KPs affected locomotor behavior in one control group and two treatment groups (AjK1 and AjK2). We discovered that AjK1 had a significant dose effect, primarily by shortening the stride length and duration of movement to reduce the sea cucumber movement distance, whereas AjK2 had little inhibitory effect at the same dose. The levels of phosphatidylethanolamine (PE), phosphatidylcholine (PC), uridine, glycine, and L-serine in the longitudinal muscle of A. japonicus treated with AjK1 differed significantly from those of the control, which may explain the observed changes in locomotor behavior. Treatment with AjK2 induced changes in aspartate levels. Our results imply that AjK1 is more likely than AjK2 to have a role in the regulation of A. japonicus locomotion.


Introduction
Neuropeptides are endogenous peptides synthesized in nerve cells. They, by and large, comprise 1-300 amino acids, and they assume significant parts in cell correspondence,

Laboratory Animals and Rearing Environment
Sea cucumbers A. japonicus used in this study were obtained from the port of Zhuwang, Laizhou, China (37 • N, 119 • E). They were put in rectangular aquariums (3 m × 2 m) filled with filtered and oxygenated seawater (TEMP 14.8 ± 0.6 ºC, SAL 29 psu, DOC > 7.5 mg/L). These sea cucumbers were fed a homemade diet consisting of sea mud plus 30% Sargassum powder. The feces and remaining feed were eliminated with a hose every day. The sea cucumbers were cultured under these conditions until used in the experiment.

Neuropeptide Synthesis
The sequences of AjK1 and AjK2 used in this study were reported by Wang et al. (2020) and were predicted in silico from A. japonicus transcriptome data, which were AGSLDCLEASCEDVERRGRQPNRNAHYRTLPF-NH 2 and SAVKNKNKSRARPPLLPF-NH 2 , individually [7]. Both AjK1 and AjK2 were verified as C-terminally amidated peptides by mass spectrometry analysis, and two cysteine residues of AjK1 were linked by a disulfide bridge [7,14]. The peptides were synthesized using a solid-phase synthesis method by APeptide Co., Ltd. (Shanghai, China). The molecular mass of AjK1 was 3659.01 g/mol, and its purity was 96.80%. The molecular mass of AjK2 was 2022.40 g/mol, and its purity was 96.61%. AjK1 and AjK2 were put away at −20 ºC before use.

Locomotion Behavior Video Capture
For the behavioral experiments, we divided 56 sea cucumbers in good condition into seven groups (n = 8 sea cucumbers/group, 99.41 ± 13.06 g). They were separated into one control group, three AjK1 treatment groups (K1), and three AjK2 treatment groups (K2). In the seawater control group (S), 0.1% (v/w) natural seawater that had been sterilized at a high temperature and in an autoclave was injected into the coelom of the animals. Sea cucumbers in the treatment groups were injected with the same volume as the S group, but with the KP solution rather than saltwater. The test concentrations of AjK1 and AjK2 were determined at 10 µM (high, H), 1 µM (medium, M), and 0.1 µM (low, L) based on Ding et al. (2019), Kato et al. (2009), and other laboratory research (unpublished data) [1,23]. AjK1 and AjK2 were dissolved in sterilized seawater and diluted to these levels. Information about the experimental groups is provided in Table 1. Each experimental sea cucumber was photographed separately in a white plastic basin (70 cm in diameter, water line 7 cm) to make it more straightforward for the analysis software to recognize individuals. A TLC200 time lapse video camera (Brinno, Taiwan, China) was mounted on a camera stand over each basin, and a 5 W bulb positioned above the basin provided brightening (Supplementary Figure S1). Before the start of the behavioral experiment, the experimental sea cucumbers were kept in the shooting environment for at least 24 h to acclimatize to the circumstance [1]. This experiment was conducted under continuous light source (12L:0D). Both before and after neuropeptide administration, the behavioral recording time was 12 h.

Data Analysis of Locomotion Behavior
EthoVision XT software (version: 10.1, Noldus Inc., Wageningen, Netherlands) was used to analyze the behavioral videos by calculating distance, moving duration, and mean velocity. Successive alternations of contraction and relaxation caused sea cucumbers to move ahead [18]. Therefore, we characterized one step as one contraction and relaxation, and stride length was the distance moved per step. By counting steps, we calculated the average stride length and stride frequency. The difference value of a behavioral parameter (∆Y) was obtained by subtracting the post-injection value from the pre-injection value, and it represented the influence of the KP on locomotor behavior of A. japonicus (∆Y > 0 inhibition, ∆Y < 0 promotion).
We assumed that the effect of the KPs on A. japonicus satisfied the first-order kinetic model [24]: We obtained the maximum effect parameter A max and decay rate constant k with this equation. We obtained the half-life τ as follows: We analyzed the locomotion data using one-way analysis of variance (ANOVA), paredsamples t-test, two-sided t-tests, and nonlinear regression analysis in SPSS 27 software (IBM, Armonk, NY, USA).

Muscle Metabolomics
For this experiment, we divided 48 healthy sea cucumbers into a control group (CM), an AjK1 treatment group (K1M), and an AjK2 treatment group (K2M), each containing 16 animals (n = 16 sea cucumbers/group, 92.11 ± 11.25 g). For the two treatment groups, the neuropeptide concentration was 10 -5 M. The injection amount of KPs was 0.1% of the body weight (v/w) of A. japonicus. After neuropeptide treatment, about 2 g of muscle tissue were taken from the body wall of each sea cucumber. In vitro tissues were cleaned with ultrapure water, dried with absorbent paper, placed in sterile cryopreservation tubes, and stored at -80 • C. Two samples were combined into one tube for metabolomics detection.
Muscle metabolomics, including metabolite extraction, LC-MS/MS analysis, and bioinformatics analysis, was carried out in collaboration with Guangzhou Genedenovo Biotechnology Co., Ltd. (Guangzhou, China). The detailed experimental procedures and methods are consistent with those of Li et al. (2019) [25]. Figure 1 shows the total distance and moving duration of A. japonicus under different treatments. Comparative with the control group, the total distance (moving duration) in the K1H group decreased after injection from 34.57 ± 17.39 m to 23.34 ± 12.84 m (from 8.36 ± 2.25 h to 5.75 ± 1.64 h), a reduction of about 30%, and the within group and between group comparisons showed significant difference; however, no apparent changes or significant differences were detected in any of the other groups. Figure 2 shows changes in inhibition of locomotion over time (0-12 h) for the AjK1 and AjK2 treatment groups at different doses (L/M/H). High doses of AjK1 had inhibition effects, especially in the first five hours after injection. The K2H group showed a reduction that lasted 2-3 h, but any influence of the other treatment groups was not obvious.   Next, we compared differences in the locomotor behavior of sea cucumbers treat with different KPs and doses. To analyze the distance, moving duration, and mean vel ity, we used one-way ANOVA for each KP, with concentration set as the factor level. F step number, average stride length, and step frequency, we used two-tailed t-tests    Next, we compared differences in the locomotor behavior of sea cucumbers treated with different KPs and doses. To analyze the distance, moving duration, and mean velocity, we used one-way ANOVA for each KP, with concentration set as the factor level. For step number, average stride length, and step frequency, we used two-tailed t-tests to Next, we compared differences in the locomotor behavior of sea cucumbers treated with different KPs and doses. To analyze the distance, moving duration, and mean velocity, we used one-way ANOVA for each KP, with concentration set as the factor level. For step number, average stride length, and step frequency, we used two-tailed t-tests to compare the distinction between K1H or K2H and S. For the first three parameters, AjK1 had a significant dose impact in the first 5 h; for the latter three parameters, the significance of K1H in the first 5 h was mainly reflected in step number and stride length and was weak in step frequency ( Figure 3). In contrast, AjK2 treatment had almost no significant effect throughout the entire experiment. compare the distinction between K1H or K2H and S. For the first three parameters, AjK1 had a significant dose impact in the first 5 h; for the latter three parameters, the significance of K1H in the first 5 h was mainly reflected in step number and stride length and was weak in step frequency ( Figure 3). In contrast, AjK2 treatment had almost no significant effect throughout the entire experiment. (d-f) The difference between the two high concentration treatment groups (K1H and K2H) and the seawater control group, which were compared using t-tests (twotailed). The significance level was set at 0.05.

Changes in Locomotion of A. japonicus after KPs Injection
Then, we analyzed contrasts in locomotor behavior of sea cucumbers treated with various KPs and dosages. To examine the distance moved, cumulative duration of movement, and mean velocity data, we involved one-way ANOVA for every KP, with concentration set as the factor level. For step number, average stride length, and step frequency data, we utilized two followed t-tests to look at the distinction between K1H or K2H and S. For the initial three parameters, AjK1 had a significant dose effect in the initial 5 h; for the last three parameters, the significance of K1H in the first 5 h was predominantly reflected in step number and stride length and was weak in step frequency ( Figure 3). Conversely, AjK2 treatment had no massive impact throughout the entire investigation.
AjK1 had a dose effect, and its effectiveness was time dependent. According to the principle of pharmacokinetics, among the steps of absorption, distribution, metabolism, and elimination, we focused on the elimination process because we had directly injected the treatment (i.e., absorption was instantaneous) [24]. We used the first-order kinetic model to fit the Amax and k, and τ was obtained by conversion. Table 2 clearly shows the dose response and pharmacokinetics of AjK1. Both high and medium concentrations of AjK1 could fit the kinetic parameters, but the low dose of AjK1 could not be fitted due to the insignificant effect. Amax of K1H and K1M were similar, but k and τ were different. The k values of K1H and K1M were about 0.25-0.3 h -1 and 0.5-0.6 h -1 , respectively, and their half-lives were about 2.5-3 h and 1.1-1.2 h, respectively. For K2H, the only parameter that could be fit by the model was stride length, and both Amax and τ were lower than those of AjK1. This indicated that at the same dose, the effect of AjK2 was far less than that of AjK1 and that AjK2 had no significant effect on the locomotor behavior of A. japonicus. (d-f) The difference between the two high concentration treatment groups (K1H and K2H) and the seawater control group, which were compared using t-tests (twotailed). The significance level was set at 0.05.
Then, we analyzed contrasts in locomotor behavior of sea cucumbers treated with various KPs and dosages. To examine the distance moved, cumulative duration of movement, and mean velocity data, we involved one-way ANOVA for every KP, with concentration set as the factor level. For step number, average stride length, and step frequency data, we utilized two followed t-tests to look at the distinction between K1H or K2H and S. For the initial three parameters, AjK1 had a significant dose effect in the initial 5 h; for the last three parameters, the significance of K1H in the first 5 h was predominantly reflected in step number and stride length and was weak in step frequency ( Figure 3). Conversely, AjK2 treatment had no massive impact throughout the entire investigation.
AjK1 had a dose effect, and its effectiveness was time dependent. According to the principle of pharmacokinetics, among the steps of absorption, distribution, metabolism, and elimination, we focused on the elimination process because we had directly injected the treatment (i.e., absorption was instantaneous) [24]. We used the first-order kinetic model to fit the A max and k, and τ was obtained by conversion. Table 2 clearly shows the dose response and pharmacokinetics of AjK1. Both high and medium concentrations of AjK1 could fit the kinetic parameters, but the low dose of AjK1 could not be fitted due to the insignificant effect. A max of K1H and K1M were similar, but k and τ were different. The k values of K1H and K1M were about 0.25-0.3 h -1 and 0.5-0.6 h -1 , respectively, and their half-lives were about 2.5-3 h and 1.1-1.2 h, respectively. For K2H, the only parameter that could be fit by the model was stride length, and both A max and τ were lower than those of AjK1. This indicated that at the same dose, the effect of AjK2 was far less than that of AjK1 and that AjK2 had no significant effect on the locomotor behavior of A. japonicus.

KPs Induced Changes in Muscle Metabolism
In this study, we used LC-MS/MS technology to identify metabolites in the muscle tissues of sea cucumbers from the three groups (CM, K1M, K2M). Metabolomics ordinarily uses QC samples for quality control analysis to obtain reliable data [26]. In our study, QC samples were well aggregated in the principal component analysis score plot, which demonstrated that the metabolomic assay results we obtained were dependable (Supplementary Figure S2). The metabolome data for the muscle samples from the three experimental groups were compared by PLS-DA in pairs to obtain the differential metabolic profiles (Figure 4). Prescient capacity (Q 2 ) values are the most recognized parameter to characterize the reliability of PLS-DA models and Q 2 > 0.5 indicated that this model predicted reliably in our study. PLS-DA score plots in both positive and negative ion modes showed relatively obvious differentiation between each of the pairs of groups, which indicated that both AjK1 and AjK2 could induce metabolism changes and that the changes evoked by the two KPs may differ.

Differential Metabolites and Metabolic Pathways under KPs Treatment
We focused on the metabolic differences between the treatment groups and the seawater group and looked at the metabolic differences between the two KP groups. Metabolites with significant changes were identified based on VIP > 1 and p < 0.05 (Supplementary  Table S1). K1M and K2M were compared with CM in two ion modes and 42 and 55 differential metabolites were detected, respectively. Treatment with AjK1 resulted in up-and down-regulation of 26 and 16 differential metabolites, respectively, while 9 and 46 upand down-regulated divergent metabolites were found in the AjK2 treatment group. The shared and specific differential metabolites in the different comparison sets are displayed in Figure 5. Only four metabolites were shared by the two treatment groups; however, the two neuropeptides regulated methyl 2-propenyl selenide in opposite directions. Overall, the two KPs had different effects. Enrichment analysis showed that the differential metabolites were involved in numerous metabolic pathways. Treatment with AjK1 and AjK2 significantly interfered with 3 and 17 of them, respectively, most of which involved the metabolism of amino acids ( Figure 6, p < 0.05).

KPs More Likely to Inhibit Locomotion of A. japonicus
The experimental results indicated that AjK1 was more likely to be involved in locomotor regulation, and AjK2 seemed not to have an effect. Although the movement pattern of A. japonicus consisted mainly of random movement, locomotor behavior was inhibited in a dose-dependent manner for the first 5 h after injection of AjK1, whereas AjK2 at the same concentration had little impact. The fundamental factors that affect movement are moving duration, step frequency, and stride length. In our study, the significance of step frequency was weak, suggesting that AjK1 was more likely to inhibit step length and moving duration and increase immobility time. The A max values of K1H and K1M were similar, but the k and τ values differed. The τ of K1H was about 2.5-3 h, which might explain the significant dose effect that occurred within 5 h. After 5 h, the effect disappeared and locomotor behavior returned to the background level. The similar A max values of K1H and K1M may indicate that 1 µM of AjK1 is enough to reach the limit of the inhibitory effect, and increasing the dose only prolongs the action time. It is worth mentioning that the stride of sea cucumbers in the K2H group showed a slight fluctuation, and enhancing the concentration of AjK2 might show the effect more clearly. In addition, there is a circadian rhythm in the locomotion of A. japonicus, and the most important factor influencing it is the change in light rhythm. Under continuous light conditions (24L:0D), there were some ups and downs, but the motor behavior rhythm of A. japonicus almost disappeared, which was consistent with the results of S group in this experiment [27]. Therefore, we chose to conduct our experiments at night using only uniform experimental light sources and a fixed experimental time, and we used ∆Y values for data analysis to minimize the effect of circadian rhythms. In other words, we were concerned with the effect of the injected neuropeptide on the locomotion of A. japonicus rather than whether the neuropeptide acted differently on A. japonicus during the day and night. Therefore, the effects of circadian rhythms in A. japonicus are not addressed in our discussion.
Most studies of locomotor behavior under the influence of KPs have focused on rats and mice, and little is known about their effects on other vertebrates and invertebrates. Kisspeptin-expressing neurons signaling in the arcuate nucleus of the hypothalamus pro-mote physical activity during the dark or active phase of adult female mice [28]. Mammalian kisspeptin-13 induces an increase in both spontaneous and exploratory locomotion in male Sprague-Dawley rats [29], whereas kisspeptin-8 (n-terminally truncated octapeptide) decreases spontaneous movement in adult male Wistar rats [30]. Kissorphin, a new peptide derived from kisspeptin-10, attenuates acute hyperkinesis and sensitization induced by morphine and ethanol in male mice [31]. Although the effects of different KPs on locomotor behavior might differ among different species, our results demonstrated that AjK1 can inhibit the locomotion of A. japonicus.
Current research results indicate that multiple neurohormones and neuropeptides are associated with the regulation of locomotor behavior. Ding et al. (2019) reported that melatonin could reduce its exercise endurance and movement efficiency [1]. In contrast, L-type SALMFamide neuropeptide enhanced the exercise endurance and movement efficiency of A. japonicus [32], and pedal peptides could enhance exercise endurance and reduce movement efficiency [17]. Kahsai et al. (2010) reported that neuropeptides expressed in the insect brain central complex are involved in the regulation of locomotor behavior [33]. They found that tachykinin could regulate the spatial orientation of Drosophila melanogaster. Cui er al. (2020) found that short neuropeptide F (sNPF) had suppressive effects on D. melanogaster locomotor activity levels, and down-regulation of sNPF increased the long-distance walking and movement velocity of female fruit flies [34]. NPF also is involved in the locomotor plasticity of locusts and attenuates phase-related locomotor activity [35]. Diaz et al. (2019) identified allatostatin-C as a novel neuropeptide that could mediate evening locomotor activity under different photoperiods in Drosophila, and Kiss et al. (2013) reported that drosulfakinin increased Drosophila larval locomotion activity and induced an escape response [36,37]. The lack of neuropeptide pigment-dispersing factor can influence the normal locomotor activity of the roundworm Caenorhabditis elegans [38]. Neuropeptides such as RYamide, DLamide, and FMRFamide regulate ciliary beat frequency and affect the swimming depth of Platynereis larvae [39]. Other reports have shown that neuropeptides such as corticotropin-releasing hormone, thyrotropin-releasing hormone, or orexin affect not only feeding behavior but also locomotor activity in goldish (Carassius auratus) [40][41][42]. Intracerebroventricular injections of orexin also can promote locomotion in mice, rats, and dogs, but it has a mild sedative effect on zebrafish (Danio rerio) and can inhibit its locomotion [43][44][45][46]. Neuropeptide S, which activates locomotion and reduces anxiety, reinstates extinguished cocaine-seeking behavior in adult male mice via corticotropin-releasing factor receptor 1 [47].
In conclusion, numerous studies have demonstrated that neuropeptides have an impact on locomotor activity. We found that AjK1 can regulate locomotor behavior by inhibiting stride length and moving duration of A. japonicus, and reduce their exercise endurance and movement efficiency. This discovery provides new evidence for the involvement of KPs in regulating locomotor behavior.

Mechanisms by Which AjK1 Inhibits Locomotor Behavior of A. japonicus
We used LC-MS/MS to qualitatively and quantitatively analyze the low-molecularweight metabolites in three groups of muscle samples of A. japonicus (CM, K1M, and K2M). The two KPs had obvious interference effects on muscle metabolites (Figure 4). The K1M and K2M groups shared only four differential metabolites, and they did not share the same differential metabolic pathways. Thus, despite the fact that both AjK1 and AjK2 were KPs, we hypothesized that their impacts on the muscle metabolism of A. japonicus were distinct. AjK1 was more likely to be involved in regulating the locomotor behavior in A. japonicus, according to behavioral data. We assume that the locomotor behavior of A. japonicus is inhibited by the significantly altered metabolites and metabolic pathways found in the K1M group. Pyrimidine metabolism, cyanoamino acid metabolism, and mineral absorption are the metabolic pathways in the longitudinal muscle that were significantly impacted by the administration of AjK1. These pathways involved differential metabolites such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), uridine, glycine, L-serine, etc.
The two most important phospholipids in eukaryotic cells are PE and PC [48]. They are the main components of cell membranes, accounting for 50% and 20-30% of total phospholipids, respectively [49,50]. Deletion of muscle-specific PC-and PE-related enzymes leads to decreased synthesis of PE and an increased PC:PE ratio, which are accompanied by decreased skeletal muscle mass and low locomotion [51][52][53]. Therefore, the PC:PE ratio is probably related to skeletal muscle growth and contraction, motor performance, and behavioral plasticity [49,54]. We found that PC18:2(9Z,12Z)/18:0), PC(24:1(15Z)/14:1(9Z)), and PE(P-18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)) were significantly up-regulated after AjK1 administration and that the PC:PE ratio was elevated. This may not only support previous research but also explain the experimental results. The regulation of phospholipids and its effect on muscle contractility may explain the change in stride length after AjK1 injection in A. japonicus. Thus, the change in the PC:PE ratio may be the underlying physiological mechanism of AjK1 inhibition of the locomotor behavior of A. japonicus.
The pyrimidine metabolic pathway not only maintains suitable pyrimidine levels but also generates bioactive intermediate metabolites [55]. For example, uridine is involved in synthetizing RNA, biofilm RNA, and biological membranes, and the concentration of uridine in human plasma is much higher than that of other purines and pyrimidines [56]. A mixture containing docosahexaenoic acid and/or eicosapentaenoic acid and uridine or its equivalents can reduce locomotor activity to help people rest well at night [57]. Honda et al. (1984) reported that after intraventricular injection of 1 nmol of uridine, locomotor and sleep-waking rhythms became irregular in rats [58]. Uridine also was found to attenuate rotation induced by methamphetamine and L-dopa in 6-OHDA-treated rats and the hyperactivity induced by acute morphine treatment in mice [59,60]. Based on the results of the influence of uridine on locomotor behavior, we speculated that the changes in locomotor performance in sea cucumbers in the AjK1 treatment group were related to uridine level.
There are 20 kinds of proteinogenic amino acids, among which glycine and serine are non-essential amino acids that can be synthesized by the organism [61]. Glycine and L-serine (one of two forms of serine) are related to several metabolic pathways. Of these pathways, cyanoamino acid metabolism and mineral absorption were significantly inhibited after AjK1 administration. Glycine is known to be inhibitory neurotransmitter in the brainstem and spinal cord [62,63]. Glycinergic interneurons can control motor rhythm generation, and mutations in glycine receptor subunits can cause human motor dysfunction [62]. Blockage of glycine receptors in the spinal cord can result in the loss of the left-right alternating locomotor pattern in newborn mice, but tonic activity increases in neonatal rats [64,65]. The detection of alternating left and right electrophysiological signals in isolated spinal nerve fibers in mice represents a marching movement, called fictive locomotion. According to Kudo et al. (2006), glycine-mediated inhibition was required for the generation of normal fictive locomotion throughout the prenatal stage of rats [66]. Down-regulation or mutations in glycine receptor subunits are also associated with major locomotor deficits in zebrafish [67]. L-serine and its associated metabolism are thought to be essential for several specific functions in the central nervous system, including cell proliferation and intracellular metabolism [68,69]. L-serine is also considered to be a free amino acid essential for exercise [70]. Furthermore, glycine is a possible source of L-serine [68]. In isolated male rats, oral administration of L-serine can decrease locomotor activity in familiar environments and exploratory behavior in novel ones [71]. Additionally, L-serine can suppress motor activity and increase the time-keeping sleeping posture in neonatal chicks under social isolation stress [69]. L-serine also inhibits the abnormal activity induced by beta-methylamino-L-alanine in D. melanogaster [72]. In conclusion, both glycine and L-serine can affect locomotor activity. Hence, the changes in L-serine and glycine levels caused by KPs may contribute to the explanation of the locomotor inhibition observed in A. japonicus in our experiment.

Possible Functions of the Neuropeptide AjK2
Results of our behavior experiment suggested that AjK2 may not participate in regulating A. japonicus locomotor activity, but it still caused changes in muscle metabolites. Among them, the alanine, aspartate, and glutamate metabolism pathway were most significantly affected by the AjK2 treatment. This pathway contains differential metabolites such as aspartate (both L-aspartate and D-aspartate). Aspartate is a nonessential amino acid, and D-aspartate participates in the regulation of sex hormones, which can stimulate the release of gonadotropin-releasing hormone and luteinizing hormone in rats [73]. Exogenous Daspartate can increase testosterone levels in male animals and affects gametogenesis [74]. Di Fiore et al. (2019) reported that the levels of D-aspartate in wild animals were directly correlated with the periodic levels of sex hormones, suggesting that this amino acid was involved in the regulation of seasonal reproduction [75]. Under the action of AjK2, the higher level of D-aspartate in the longitudinal muscle of A. japonicus showed significant changes, which may confirm the premise of Wang et al. (2020) that KPs are related to the seasonal reproduction of A. japonicus [7].

Conclusions
Results of our behavior experiment suggested that AjK1 was more likely to regulate locomotor behavior of A. japonicus than AjK2, and that AjK2 seemed to have little effect on it. After the intraperitoneal injection of AjK1, locomotor activity was inhibited, and exercise endurance and movement efficiency decreased. Muscle physiology analysis revealed changes in uridine, glycine, and L-serine levels after AjK1 administration, which may be a potential mechanism responsible for the inhibition of locomotor behavior. Our results suggested that AjK2 is more likely to be related to seasonal reproduction. Further enhancement of AjK2 concentration may be needed to observe its effects on locomotion. More in-depth studies of pharmacokinetics and the underlying biological mechanisms are needed to fill the gaps in our understanding of kisspeptin neuropeptides in invertebrates.  Institutional Review Board Statement: All experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Ethical Committee and the protocols were approved by the Committee on the Ethics of Animal Experiments of the Institute of Oceanology, Chinese Academy of Sciences, Shandong, and China. All efforts were taken to minimize the suffering of the animals. The animals were anesthetized with magnesium sulfate before dissection. No licenses or permits were required for this study.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article. Further information is available upon request from the corresponding author.